MOLECULAR CHARACTERIZATION OF TWO

VITOR BATISTA PINTO
MOLECULAR CHARACTERIZATION OF TWO BEGOMOVIRUSES
INFECTING Pavonia sp. (MALVACEAE), AND ANALYSIS OF THE INTRAHOST EVOLUTION OF Tomato severe rugose virus (ToSRV)
Dissertação apresentada à Universidade Federal
de Viçosa, como parte das exigências do
Programa de Pós-Graduação em Genética e
Melhoramento, para obtenção do título de
Magister Scientiae.
VIÇOSA
MINAS GERAIS - BRASIL
2015
Aos meus pais Milton e Eliana,
dedico.
ii
AGRADECIMENTOS
A Deus, por sempre iluminar meus caminhos e permitir mais essa conquista
em minha vida;
Aos meus pais, Milton e Eliana, pelo constante apoio, amor, carinho e por
sempre acreditarem no meu potencial;
À minha irmã Renata pela amizade e incentivo;
Ao Professor Murilo Zerbini, pela orientação, amizade, paciência e dedicação
para realização deste trabalho;
À Universidade Federal de Viçosa (UFV) por proporcionar a realização do
mestrado em Genética e Melhoramento;
Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq) pelo auxílio financeiro;
Aos funcionários da Pós-Graduação em Genética e Melhoramento pela
paciência e disponibilidade todas as vezes nas quais precisei;
Aos amigos da Pós-Graduação em Genética e Melhoramento, especialmente à
Isadora, sem você meu mestrado não teria sido o mesmo;
Aos amigos do Laboratório de Virologia Vegetal Molecular, Josiane, André,
Hermano, Márcio, Igor, César, Larissa, Letícia, Flávia, Fernanda, Murilo, Talita,
Priscila, Patrícia e especialmente à Camila, Angélica e João pela sincera amizade,
apoio e momentos de descontração;
Ao Cleysinho e Márcio pela valiosa colaboração na execução deste trabalho;
Ao servidor Pedro M. P. Vidigal e ao Núcleo de Análises de Biomoléculas
(NuBioMol) da Universidade Federal de Viçosa pelo suporte na análise dos dados
gerados;
iii
Aos familiares que me apoiaram e sempre deram palavras de incentivo que
confortavam nos momentos mais difíceis;
Aos amigos Mateus, Mayara, Luiza, Daiana, Luiz Felipe, Sil, Paulo, Daniel,
Úrsula, Christiane, Adriana, Marília, Carla, Carol M., Rizia, Gustavo e Rosane que,
mesmo distantes, sempre se fazem presentes e a todos os amigos que conquistei nesta
etapa, em especial ao Luan, Carol H., Thaís, Rodrigo, Vinícius e Marcone, pelas
risadas, histórias, festas e por me aguentarem todo esse tempo em Viçosa;
Aos companheiros de república, Michel, Pedro e Vinícius, pela convivência,
compreensão e paciência;
A todos que contribuíram direta ou indiretamente para conclusão deste
trabalho.
iv
BIOGRAFIA
VITOR BATISTA PINTO, filho de Milton Antônio Caetano Pinto e Eliana
Pereira Batista Pinto, nasceu em Sabará, Minas Gerais, no dia 19 de novembro de
1988.
Realizou o ensino médio e o curso técnico em Química Industrial no Centro
Federal de Educação Tecnológica de Minas Gerais (CEFET-MG), sendo concluído
em 2006. Graduou-se em Agronomia pela Universidade Federal de Minas Gerais
(UFMG), na cidade de Montes Claros, Minas Gerais, em julho de 2013. Ingressou no
mês seguinte no curso de pós-graduação em Genética e Melhoramento em nível de
Mestrado na Universidade Federal de Viçosa e, em julho de 2015, submeteu-se à
defesa de dissertação.
v
SUMÁRIO
Abstract ......................................................................................................................... vii
Resumo .......................................................................................................................... .ix
Introduction .................................................................................................................. 1
Chapter 1. Two novel begomoviruses infecting the malvaceous weed
Pavonia sp. in Brazil..................................................................................................... 12
Abstract ..................................................................................................................... 13
Acknowledgments ..................................................................................................... 17
References ................................................................................................................. 17
Tables, figures and supplementary files .................................................................... 20
Chapter 2. Intra-host evolution of Tomato severe rugose virus (ToSRV)................ 31
Abstract ..................................................................................................................... 32
Introduction ............................................................................................................... 33
Material and Methods ................................................................................................ 36
Results ....................................................................................................................... 39
Discussion ................................................................................................................. 42
References ................................................................................................................. 46
Tables, figures and supplementary files .................................................................... 50
vi
ABSTRACT
PINTO, Vitor Batista, M.Sc., Universidade Federal de Viçosa, July, 2015.
Molecular characterization of two begomoviruses infecting Pavonia sp.
(Malvaceae), and analysis of the intra-host evolution of Tomato severe rugose
virus (ToSRV). Advisor: Francisco Murilo Zerbini Júnior.
The family Geminiviridae is comprised of viruses with a circular, single-stranded
DNA genome encapsidated in twinned icosahedral particles. The viruses in the genus
Begomovirus are transmitted by the whitefly Bemisia tabaci to dicot plants.
Begomoviruses have mutation and nucleotide substitution rates similar to those
reported for RNA viruses, and a high frequency of recombination. Due to their rapid
evolutionary process, new begomovirus species are often found in the field. This
study aimed to perform the molecular characterization of two begomovirus species
infecting Pavonia sp. (Malvaceae), and to follow and quantify the evolution of
Tomato severe rugose virus (ToSRV) in a cultivated and a non-cultivated host. Two
begomoviruses were isolates from Pavonia sp. plants collected in the municipalities
of Albuquerque and Corumbá, Mato Groso do Sul, Brazil. Sequence comparisons
and phylogenetic analysis showed that these were two novels species, with the
typical features of bipartite, New World begomoviruses. The names Pavonia mosaic
virus (PavMV) and Pavonia yellow mosaic virus (PavYMV) were proposed for the
two new species. In the study to evaluate the evolutionary dynamics of ToSRV,
tomato and Nicandra physaloides plants were inoculated via biobalistics with an
infectious clone of ToSRV and maintained in a greenhouse. Total DNA was extrated
from leaves collected at 30, 75 and 120 days after inoculation, and was sequenced in
the Illumina HiSeq 2000 platform. The DNA libraries from each of the two hosts
were submitted to quality control analysis with FastQC software. The genome
vii
assembly was performed with the program Geneious using the infectious clone as
reference. Both genomic components of ToSRV showed substitution rates similar to
those of RNA viruses: 3.06 x 10-3 and 2.03 x 10-3 sub/site/year for the DNA-A and
DNA-B, respectively, in N. physaloides, and 1.38 x 10-3 and 8.68 x 10-4 sub/site/year
for the DNA-A and DNA-B, respectively, in tomato. Substitution rates in the range
of those already described for other begomoviruses were found also for the CP, Rep,
MP and NSP genes in both hosts. We quantified synonymous and non-synonymous
substitutions, transversions and transitions, as well as deletions and insertions in the
CP, Rep, MP and NSP genes. A decrease in the number of variable sites was
observed during the course of the experiment, with a corresponding increase in the
number of identical sites to the reference genome. Suppression of the stop codons of
the MP and NSP genes was observed in the N. physaloides libraries, suggesting an
adaptive strategy. Determination of Shannon entropy indicated mutation hotspots in
the N-terminal region of the Rep gene, the intergenic common region in the DNA-A
and DNA-B (CR-A and CR-B, respectively) and the long intergenic region between
the MP and NSP genes in the DNA-B (LIR-B). Overall, the results indicate that
ToSRV evolves as a quasispecies, with a high degree of genetic variability which
could be partly responsible for its prevalence in the field.
viii
RESUMO
PINTO, Vitor Batista, M.Sc., Universidade Federal de Viçosa, julho de 2015.
Caracterização molecular de dois begomovírus infectando Pavonia sp.
(Malvaceae), e análise da evolução intra-hospedeiro do Tomato severe rugose
virus (ToSRV). Orientador: Francisco Murilo Zerbini Júnior.
A família Geminiviridae é composta por vírus com genoma de DNA circular de fita
simples, encapsidado em partículas icosaédricas geminadas. Os vírus pertencentes ao
gênero Begomovirus são transmitidos pela mosca-branca Bemisia tabaci a plantas
dicotiledôneas. Os begomovírus apresentam taxas de mutação e substituição
nucleotídica semelhantes às relatadas para vírus de RNA, e uma elevada taxa de
recombinação. Devido ao seu rápido processo evolutivo, novas espécies de
begomovírus são frequentemente encontradas no campo. Este trabalho teve como
objetivos realizar a caracterização molecular de duas espécies de begomovírus
infectando Pavonia sp. (Malvaceae), e quantificar a evolução do Tomato severe
rugose virus (ToSRV) em um hospedeiro cultivado e um não-cultivado. Dois
begomovírus foram isolados de plantas de Pavonia sp. coletadas nos municípios de
Albuquerque e Corumbá, Mato Grosso do Sul. Comparação de sequências e análise
filogenética indicaram tratar-se de duas novas espécies com as características de
begomovírus bissegmentados das Américas. Foram propostos os nomes Pavonia
mosaic virus (PavMV) e Pavonia yellow mosaic virus (PavYMV). No estudo para
avaliar a dinâmica evolutiva do ToSRV, plantas de tomateiro e Nicandra physaloides
foram inoculadas via biobalística com clone infeccioso do ToSRV e mantidas em
casa-de-vegetação. DNA total foi extraído de folhas coletadas aos 30, 75 e 120 dias
após a inoculação, e submetido a sequenciamento na plataforma Illumina HiSeq
2000. As bibliotecas de DNA foram submetidas a análise de qualidade no software
ix
FastQC. O alinhamento dos reads foi realizado no software Geneious, utilizando o
clone infeccioso como sequência referência. Ambos os componentes genômicos do
ToSRV apresentam taxa de substituição semelhantes a de vírus de RNA: 3,06 x 10-3
e 2,03 x 10-3 subst/sítio/ano para o DNA-A e DNA-B, respectivamente, em N.
physaloides, e 1,38 x 10-3 e 8,68 x 10-4 subst/sítio/ano para o DNA-A e DNA-B,
respectivamente, em tomateiro. Valores de taxa de substituição similares aos já
descritos para begomovírus foram encontrados também para os genes CP, Rep, MP e
NSP em ambos os hospedeiros. Foram quantificadas substituições sinônimas e nãosinônimas, transversões e transições, além de deleções e inserções nos genes CP,
Rep, MP e NSP. Foi observado um decréscimo no número de variações ao longo do
tempo e consequente aumento do número de sítios idênticos ao genoma referência.
Nas bibliotecas provenientes de N. physaloides foi observada a supressão dos códons
de terminação dos genes MP e NSP em todo decorrer da infecção, sugerindo uma
estratégia adaptativa. O cálculo da entropia de Shannon identificou como hotspots de
mutação a região N-terminal do gene Rep, as regiões comuns no DNA-A e DNA-B
(CR-A e CR-B, respectivamente) e a região intergênica longa entre os genes MP e
NSP no DNA-B (LIR-B). Estes dados sugerem que o ToSRV evolui como
quasispecies, apresentando elevada variabilidade genética, o que poderia explicar em
parte sua prevalência no campo.
x
INTRODUCTION
Various agricultural systems in different regions of the world have suffered
serious economic constraints due to geminivirus infection. Some factors, including
the emergence of new viral species/variants, emergence of more efficient vector
populations, climate change, changes in production systems, unchecked transit of
infected material and introduction of susceptible varieties, individually or together,
have contributed to the increased incidence and severity of infection by
geminiviruses (Varma and Malathi, 2003).
The Geminiviridae family is comprised of viruses with a circular, singlestranded DNA genome encapsidated in twinned icosahedral particles. Members of
this family can infect monocot or dicot plants and are widespread in all tropical and
subtropical regions of the world, causing severe diseases in many economically
relevant crops such as bean, cassava, cotton, maize, pepper and tomato (Moffat,
1999; Shepherd et al., 2010; Navas-Castillo et al., 2011). Based on the type of insect
vector, host range, genomic organization and phylogenetic relationships, the
geminiviruses are classified into seven genera: Begomovirus, Becurtovirus,
Curtovirus, Eragrovirus, Mastrevirus, Topocuvirus and Turncurtovirus (Brown et
al., 2012; Varsani et al., 2014). The begomoviruses have one or two genomic
components, are transmitted by the whitefly Bemisia tabaci and infect dicot plants.
Begomoviruses can be divided into two groups: Old World (Eastern
hemisphere: Europe, Africa, Asia and Oceania) and New World (Western
hemisphere: the Americas) (Rybicki, 1994; Padidam et al., 1999a; Paximadis et al.,
1999). Most Begomoviruses from the New World have two genomic components
known as DNA-A and DNA-B (Fauquet et al., 2005). Tomato leaf deformation virus
1
(ToLDeV), which has only a single, DNA-A-like component, was the first
monopartite begomovirus reported to naturally occur in the New World (Melgarejo
et al., 2013). The two components of bipartite begomoviruses do not share significant
sequence identity, except for a region with approximately 200 nt known as the
common region (CR), which includes the origin of replication (Hanley-Bowdoin et
al., 1999). Old World (OW) begomoviruses can be either mono- or bipartite.
Monopartite begomoviruses have a genomic component which is homologous to the
DNA-A of bipartite begomoviruses (Padidam et al., 1996; Mansoor et al., 2003).
Populations of geminiviruses, including begomoviruses, have a high degree
of genetic variability, comparable to viruses with RNA genomes (Ge et al., 2007;
Prasanna et al., 2010; Rocha et al., 2013). The main sources of genetic variability in
plant viruses are mutation, recombination and pseudo-recombination (Monci et al.,
2002; García-Arenal et al., 2003; Seal et al., 2006). Frequent recombination events
(Padidam et al., 1999b; Rocha et al., 2013), the occurrence of pseudo-recombination
between viruses with bipartite genomes (Gilbertson et al., 1993; Andrade et al.,
2006) and high mutation and nucleotide substitution rates (Duffy et al., 2008; Duffy
and Holmes, 2009) are the main factors that promote the high variability observed
for begomoviruses.
Mutation is the primary mechanism for generation of variability, with natural
selection, recombination, genetic drift and gene flow acting to mold the genetic
structure of the population (Duffy et al., 2008). The mutation rates and nucleotide
substitution rates observed for geminiviruses are similar to those calculated for RNA
viruses, despite the expectation that they would be lower since geminiviruses use the
host's proof-reading replication machinery, which in theory would increase the
fidelity of replication (Duffy et al., 2008). There is evidence that the rapid evolution
2
of geminiviruses is at least partly directed by mutational processes that act
specifically on ssDNA (Harkins et al., 2009). Studies in bacterial and animal systems
have indicated that substitutions rates of dsDNA and ssDNA viruses differ
significantly (Duffy et al., 2008).
Recombination is a very common event in geminiviruses (Padidam et al.,
1999b; Lefeuvre et al., 2009) and seems to contribute greatly to their genetic
variability, increasing their evolutionary potential and local adaptation (Harrison and
Robinson, 1999; Padidam et al., 1999b; Berrie et al., 2001; Monci et al., 2002; Lima
et al., 2013; Rocha et al., 2013). Knowledge on the existence and frequency of
recombination in a virus population may further advance the understanding of which
genes are interchanged. This information is important, for example, to maximize the
durability of genetic resistance, since new recombinant variants may have an
increased ability to infect previously resistant genotypes (Monci et al., 2002;
Awadalla, 2003; Sattar et al., 2013). Indeed, recombination events have been directly
implicated in the emergence of new begomovirus diseases and epidemics (Zhou et
al., 1997; Pita et al., 2001), including the devastating epidemic of cassava mosaic in
Uganda and neighboring countries, caused by a recombinant isolate of East African
cassava mosaic virus (Zhou et al., 1997; Pita et al., 2001), the epidemics of tomato
leaf curl disease in Spain, with the emergence of the recombinant species Tomato
yellow leaf curl Malaga virus and Tomato yellow leaf curl Axarquia virus (Monci et
al., 2002; García-Andrés et al., 2006; García-Andrés et al., 2007a; García-Andrés et
al., 2007b), and the epidemics of cotton leaf curl disease in Pakistan caused by a
complex of recombinant Cotton leaf curl virus isolates (Zhou et al., 1998; Idris and
Brown, 2002).
3
The existence of two genomic components in many begomoviruses promotes
an alternative mechanism for the exchange of genetic material, known as pseudorecombination (also known as reassortment): the exchange of genomic components
between two distinct viruses (more often strains of the same species, but sometimes
between closely related species) without the need for intermolecular recombination
(Gilbertson et al., 1993; Sung and Coutts, 1995; Andrade et al., 2006; revised by
Rojas et al., 2005). The viability of pseudo-recombinants indicates that factors
involved in replication and movement can be interchangeable. On the other hand, the
often observed asymmetry of reciprocal pseudo-recombinants indicates that this is a
complex phenomenon involving interactions among multiple viral and host factors
(Hill et al., 1998).
Typically, in genetic variability studies, the complete genome of
begomoviruses is cloned from rolling circle amplification (RCA) or PCR products
(Briddon et al., 2000; Inoue-Nagata et al., 2004), followed by conventional
sequencing. As informative as these studies have been, they are limited by the
impossibility of cloning every single variant present in an infected plant - inevitably,
only the most prevalent variants will be cloned. Thus, even the most complete studies
provide only a rough estimate of the true genetic variability of the viral population,
and may grossly underestimate the presence of minor (less fit) variants which could
become prevalent after a genetic bottleneck, or after horizontal transfer.
Recently, a study used a new approach, RCA followed by next generation
sequencing (NGS), to evaluate the diversity of a population of a begomovirus and
associated DNA satellites in naturally infected tomato and okra plants (Idris et al.,
2014). The authenticity of the sequences and reproducibility of the approach was
validated by comparing the results with those obtained by cloning and Sanger
4
sequencing. This was the first report of NGS implementation to explore the diversity
and identify begomovirus-satellite complexes directly out of naturally infected
plants, optimizing the exploration of diversity and populational structure
independently of viral abundance (Idris et al., 2014).
NGS was used also to determinate the nucleotide substitution rate of a
potyvirus. Dunham et al. (2014) determined the extension and pattern of genetic
diversity of Zucchini yellow mosaic virus (ZYMV) by sequencing 23 leaves that
grew sequentially along a single Curcubita pepo subsp. texana vine. The authors
verified that systemic movement is characterized by sequential bottlenecks in the
ZYMV population, although not enough to reduce the population to a single virion
since multiple variants were consistently transmitted between the leaves. Moreover,
the authors examined the fixation of mutations that resulted in a conformational
change in the CI protein, suggesting that these mutations may confer a selective
advantage related to systemic movement of the virus in C. pepo.
This study aimed to perform the molecular characterization of two novel
begomoviruses infecting non-cultivated plants in Brazil, and follow and quantify the
evolution of the begomovirus Tomato severe rugose virus (ToSRV) in a cultivated
host (tomato) and in a non-cultivated host (Nicandra physaloides) using NGS.
5
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11
CHAPTER 1
TWO NOVEL BEGOMOVIRUSES INFECTING THE MALVACEOUS
WEED Pavonia SP. IN BRAZIL
Pinto, V.B., Silva, J.P., Fiallo-Olivé, E., Navas-Castillo, J., Zerbini, F.M. Two novel
begomoviruses infecting the malvaceous weed Pavonia sp. in Brazil. Archives of
Virology, submitted.
12
Two novel begomoviruses infecting the malvaceous weed Pavonia sp. in Brazil
Vitor Batista Pinto1,2, João Paulo Silva1,2, Elvira Fiallo-Olivé2,3, Jésus NavasCastillo2,3, Francisco Murilo Zerbini1,2
¹Dep. de Fitopatologia, ²National Institute for Plant-Pest Interactions, Universidade
Federal de Viçosa, Viçosa, MG, 36570-900, Brazil; ³Instituto de Hortofruticultura
Subtropical y Mediterránea "La Mayora", Universidad de Málaga-Consejo Superior
de Investigaciones Científicas (IHSM-UMA-CSIC), 29750 Algarrobo-Costa,
Málaga, Spain
Abstract
Begomoviruses are whitefly-transmitted, single-stranded DNA viruses that are often
associated with non-cultivated plants. Here, we report the detection of members of
two new begomovirus species in Pavonia sp. (Malvaceae). Sequence comparisons
and phylogenetic analysis have shown that these novel species are related to New
World begomoviruses. The nucleotide sequences of the DNA-A of both viruses had
the greatest identity with Abutilon mosaic Bolivia virus (AbMBoV). Based on
symptoms observed in the field and considering the host, we proposed the names
Pavonia mosaic virus (PavMV) and Pavonia yellow mosaic virus (PavYMV) for
these two new begomoviruses.
The genus Begomovirus (family Geminiviridae) includes species that infect
dicotyledoneous plants and whose genomes are composed of one or two molecules
13
of circular, single-stranded DNA [1]. In nature, these viruses are spread by the
Bemisia tabaci sibling species group (Hemiptera: Aleyrodidae) and its distribution is
related to the spread of the vector [2, 3]. The species B. tabaci Middle East-Asia
Minor 1 (MEAM1) is highly efficient in transmitting begomoviruses and has a wide
host range [4, 5]. Begomoviruses indigenous to the New World are typically
bipartite, except for Tomato leaf deformation virus, recently described in Peru and
Ecuador [6]. The two genomic components, referred to as DNA-A and DNA-B, are
of similar size (2.5-2.7 kb). The DNA-A contains five open reading frames (ORFs)
which encode proteins with functions in viral replication (Rep, Ren), suppression of
host defenses (Trap) and particle formation (CP), while the two ORFs in the DNA-B
enconde a movement protein (MP) and a nuclear shuttle protein which is also
involved in suppression of host defenses (NSP) [7, 8]. Begomoviruses cause severe
diseases in economically important crops throughout the tropics and subtropics, and
are also frequently associated with non-cultivated plants [5]. Malvaceous plants are
one of the largest natural begomovirus reservoirs in the Americas [9-17]. The genus
Pavonia, probably the largest within the family Malvaceae, is represented by 250
species [18]. Many species are used as ornamentals and some have potential for the
cellulose industry by having cellulose without contaminants [18].
As part of an ongoing effort to assess begomovirus diversity and the
emergence of novel species, symptomatic Pavonia sp. plants were collected in the
cities of Albuquerque (S19º23'49.7'', W57º25'29.5''; sample #51) and Corumbá
(S19°11'05.8'', W057°31'54.1''; sample #40) in Mato Grosso do Sul state, Brazil, in
September 2014 (Suppl. Fig. S1). Total DNA was extracted [19], and the presence of
a begomovirus was confirmed by rolling-circle amplification (RCA) [20]. Full-length
genomic components were cloned into pBLUESCRIPT KS+ (Stratagene) after
14
monomerization with the restriction enzymes ApaI, EcoRI, and SacI and were
completely sequenced by primer walking (Macrogen, Inc., Seoul, South Korea). The
assembly of nucleotide sequences was carried out using Geneious version 8.0.5.
Pairwise sequence comparisons were performed using Sequence Demarcation Tool
(SDT) v.1.2 [21]. Multiple sequence alignments were obtained using the MUSCLE
algorithm implemented in MEGA6 [22]. Phylogenetic trees were constructed using
Bayesian inference performed with MrBayes v.3 [23], using the General Time
Reversible (GTR) nucleotide substitution model selected by MrModeltest v.2.2 [24]
in the Akaike Information Criterion (AIC). The analysis was run for 10 million
generations, excluding the first 2,000,000 generations as burn-in. The trees were
visualized
in
FigTree
v.1.3.1
(http://tree.bio.ed.ac.uk/software/figtree).
Recombination analysis was performed with Recombination Detection Program
(RDP) v.4.5.1 [25] using default settings and a Bonferroni-corrected P-value cutoff
of 0.05. Only the recombination events detected by more than four of the seven tests
implemented in RDP were considered to be reliable.
DNA-A and -B components were cloned for the two Pavonia samples. All
four components have the conserved nonanucleotide that contains the origin of
replication (5'-TAATATTAC-3'). That the A and B components from each sample
are cognate components of a bipartite begomovirus is indicated by their identical
iteron sequences (GGTG for the components from sample #40; GGGG for the
components from sample #51) and by the digestion of the RCA products from each
sample with a 4-base cutter restriction enzyme, which indicates that they were the
only two DNA components present in each sample (data not shown). All four DNA
components have the typical organization of New World, bipartite begomoviruses,
with five ORFs in the DNA-A and two ORFs in the DNA-B. Pairwise sequence
15
analysis of the DNA-A indicated that the two isolates, named BR-Cor40-14 and BRAlb51-14, have 87% nucleotide sequence identity with each other and <82% identity
with previously described begomoviruses (Table 1). The two DNA-B components
are 89% identical and have <79% identity with other begomoviruses. The most
closely related species is Abutilon mosaic Bolivia virus (AbMBoV) for both
components (Table 1). Based on the begomovirus species demarcation criteria
recently updated by the Geminiviridae study group of the ICTV [1], each isolate
comprises a new begomovirus species for which we propose the names Pavonia
yellow mosaic virus (PavYMV) (BR-Cor40-14) and Pavonia mosaic virus (PavMV)
(BR-Alb51-14).
Pairwise sequence comparisons based on the deduced amino acid sequences
of each viral protein indicated that the CP and Ren are the most conserved proteins in
both species, with 78-85% and 79-89% identity with other begomoviruses,
respectively, and Rep and NSP the least conserved, both with <81% identity (Suppl.
Tables S2-S7).
Phylogenetic analysis based on both the complete DNA-A and DNA-B
sequences indicate that PavMV and PavYMV are most closely related to AbMBoV
(Fig. 1; Suppl. Fig. S2).
Recombination events were detected in the genomes of both viruses. The
events involved two genomic regions for PavYMV (nt 674-1226 and 1994-2198 in
the CP and Rep genes, respectively) and three for PavMV (nt 355-408 in the CP
gene; nt 2049-2197 and 2198-2433 in the Rep gene). Malvaceous-infecting
begomoviruses from the same phylogenetic clade (AbMBoV, SiBoV) were identified
as putative parents. These results indicate that, as for the vast majority of
16
begomoviruses, recombination is involved in the evolution of both PavMV and
PavYMV.
The presence of whitefly-transmitted viruses in non-cultivated malvaceous
hosts, causing the so-called "infectious chlorosis", has been reported in Brazil since
the 1940's [26]. However, it was only with the recent development of sequenceunbiased detection tools that the incredible species diversity of these viruses started
to unravel [14, 17, 27, 28]. It is curious that severe epidemics of begomoviruses in
malvaceous crops such as cotton and okra have not been reported in Brazil, unlike in
other regions such as the Indian Subcontinent and West Africa [29, 30]. This could
be related to the preference of local whitefly populations for other hosts.
Acknowledgments
This work was funded by the Science Without Borders program of CNPq (grant
401838/2013-7 to FMZ and JNC) and by FAPEMIG (grant CAG-APQ-02037-13 to
FMZ). JNC was the recipient of a Special Visiting Scientist fellowship under the
SWB program. EFO was the recipient of a Visiting Scientist fellowship from
FAPEMIG.
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Table 1 - Percent identities between the complete DNA-A (above the diagonal) and DNA-B (below the diagonal) nucleotide sequences of the
two new begomoviruses species detected in Pavonia sp. (Pavonia mosaic virus, PavMV, and Pavonia yellow mosaic virus, PavYMV) with the
most closely related begomoviruses.
TGMVa
ToSRV
ToMoLCV
ToLDV
ToYSV
SiMBoV1
SiBrV
SimMV
SiMoV
SiYNV
AbMBoV
BGMV
PavMVc
PavYMVd
TGMV
-71
66
-b
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70
70
68
69
68
68
69
ToSRV
82
-68
70
70
69
76
72
69
70
71
ToMoLCV
77
76
-67
67
67
66
67
67
66
67
ToLDV
78
80
77
--
ToYSV
79
80
77
84
-70
69
73
68
68
70
70
SiMBoV1
81
78
79
80
81
-70
73
73
69
72
72
SiBrV
76
77
75
82
80
77
-69
70
69
73
73
SimMV
78
79
76
84
83
81
80
-70
69
70
70
SiMoV
78
78
76
83
88
80
80
83
--
SiYNV
79
79
77
84
89
82
81
83
88
--
AbMBoV
78
78
75
79
79
78
75
76
78
79
-67
78
79
BGMV
75
80
74
79
78
76
80
76
76
78
76
-68
69
PavMV
76
76
76
78
78
79
78
76
76
78
82
77
-89
PavYMV
75
75
76
78
79
78
75
76
77
79
82
74
87
--
a
Access numbers as in Fig 1.
Not done (sequences not available)
c
Isolate BR-Alb51-14
d
Isolate BR-Cor40-14
b
20
Figure 1 - Phylogenetic tree based on the complete DNA-A sequences of the two
new begomovirus species detected in Pavonia sp. (Pavonia mosaic virus, PavMV,
and Pavonia yellow mosaic virus, PavYMV), plus additional sequences from New
World begomoviruses. The tree was constructed by Bayesian inference using the
GTR nucleotide substitution model. Numbers at the nodes indicate Bayesian
posterior probabilities. East African cassava mosaic virus (EACMV, an Old World
begomovirus) was used as an outgroup. AbMBoV, Abutilon mosaic Bolivia virus;
BGMV, Bean golden mosaic virus; CabLCJV, Cabbage leaf curl Jamaica virus;
CoYSV, Corchorus yellow spot virus; MaYSV, Macroptilium yellow spot virus;
OYMOIV, Okra yellow mottle Iguala virus; PYMTV, Potato yellow mosaic
Trinidad virus; SiBrV, Sida Brazil virus; SiCmMV, Sida commom mosaic virus;
SiMAV, Sida mosaic Alagoas virus; SiMBoV1, Sida mosaic Bolivia virus 1;
SimMV, Sida micrantha mosaic virus; SiMoAV, Sida mottle Alagoas virus; SiMoV,
Sida mottle virus; SiYMoV, Sida yellow mottle virus; SiYNV, Sida yellow net virus;
TGMV, Tomato golden mosaic virus; ToDLV, Tomato dwarf leaf virus; ToLDV,
Tomato leaf distortion virus; ToMoLCV, Tomato mottle leaf curl virus; ToSRV,
Tomato severe rugose virus; ToYSV, Tomato yellow spot virus; ToYVSV, Tomato
yellow vein streak virus.
21
Supplementary Figure S1 - Symptoms of mosaic and bright yellow mosaic in the
two Pavonia sp. samples collected in Albuquerque (left) and Corumbá (right).
22
Supplementary Table S1 - Percent identities between the deduced amino acid sequences of the Rep (AC1) protein encoded by the DNA-A of
the two new begomovirus species detected in Pavonia sp. (Pavonia mosaic virus, PavMV, and Pavonia yellow mosaic virus, PavYMV) and the
most closely related viruses.
TGMV
ToSRV
ToMoLCV
ToLDV
ToYSV
SiMBoV1
SiBrV
SimMV
SiMoV
SiYNV
AbMBoV
BGMV
PavMV
PavMYV
TGMV
ToSRV
ToMoLCV
ToLDV
ToYSV
SiMBoV1
SiBrV
SimMV
SiMoV
SiYNV
AbMBoV
BGMV
PavMV
--
82
--
75
75
--
79
79
74
--
82
81
76
81
--
81
80
79
79
83
--
73
74
72
78
75
75
--
80
80
75
79
80
83
75
--
78
78
75
78
86
81
75
78
--
79
78
75
81
87
85
76
80
87
--
79
79
74
79
79
81
74
75
78
79
--
74
79
73
79
76
76
81
76
74
76
76
--
76
74
74
75
75
77
77
74
74
76
81
77
--
PavMYV
75
75
76
76
77
77
72
73
76
79
80
72
82
--
23
Supplementary Table S2 - Percent identities between the deduced amino acid sequences of the Trap (AC2) protein encoded by the DNA-A of
the two new begomovirus species detected in Pavonia sp. (Pavonia mosaic virus, PavMV, and Pavonia yellow mosaic virus, PavYMV) and the
most closely related viruses.
TGMV
ToSRV
ToMoLCV
ToLDV
ToYSV
SiMBoV1
SiBrV
SimMV
SiMoV
SiYNV
AbMBoV
BGMV
PavMV
PavYMV
TGMV
ToSRV
ToMoLCV
ToLDV
ToYSV
SiMBoV1
SiBrV
SimMV
SiMoV
SiYNV
AbMBoV
BGMV
PavMV
PavYMV
--
81
--
79
78
--
79
80
78
--
80
82
79
86
--
86
78
81
82
85
--
77
79
77
84
83
76
--
80
81
80
88
84
80
84
--
82
81
79
85
93
84
82
87
--
81
82
78
86
92
84
82
85
93
--
82
80
79
82
87
85
78
81
84
85
--
77
83
74
81
81
78
83
81
80
82
79
--
80
77
81
81
83
84
79
78
81
82
89
79
--
79
78
81
81
85
84
78
81
83
83
88
79
96
--
24
Supplementary Table S3 - Percent identities between the deduced amino acid sequences of the Ren (AC3) protein encoded by the DNA-A of
the two new begomovirus species detected in Pavonia sp. (Pavonia mosaic virus, PavMV, and Pavonia yellow mosaic virus, PavYMV) and the
most closely related viruses.
TGMV
ToSRV
ToMoLCV
ToLDV
ToYSV
SiMBoV1
SiBrV
SimMV
SiMoV
SiYNV
AbMBoV
BGMV
PavMV
PavYMV
TGMV
ToSRV
ToMoLCV
ToLDV
ToYSV
SiMBoV1
SiBrV
SimMV
SiMoV
SiYNV
AbMBoV
BGMV
PavMV
--
83
--
81
83
--
82
82
82
--
82
85
83
87
--
87
84
82
83
84
--
80
82
79
85
87
81
--
81
82
81
87
86
81
83
--
81
82
80
85
90
80
84
87
--
82
84
81
86
93
83
85
87
93
--
81
81
82
83
84
83
81
82
82
84
--
80
84
78
82
83
81
83
82
82
82
81
--
81
81
82
81
83
83
83
80
80
82
89
81
--
PavYMV
81
83
81
83
85
83
81
81
82
84
88
80
94
--
25
Supplementary Table S4 - Percent identities between the deduced amino acid sequences of the AC4 protein encoded by the DNA-A of the two
new begomovirus species detected in Pavonia sp. (Pavonia mosaic virus, PavMV, and Pavonia yellow mosaic virus, PavYMV) and the most
closely related viruses.
TGMV
ToSRV
ToMoLCV
ToLDV
ToYSV
SiMBoV1
SiBrV
SimMV
SiMoV
SiYNV
AbMBoV
BGMV
PavMV
PavYMV
TGMV
ToSRV
ToMoLCV
ToLDV
ToYSV
SiMBoV1
SiBrV
SimMV
SiMoV
SiYNV
AbMBoV
BGMV
PavMV
--
86
--
72
72
--
84
83
77
--
87
85
73
87
--
82
83
74
86
86
--
67
70
68
74
71
73
--
84
86
74
88
87
91
71
--
81
78
70
79
84
79
70
78
--
83
81
76
87
88
86
72
86
87
--
80
78
71
80
79
80
69
77
77
81
--
67
69
69
74
71
72
88
74
69
76
67
--
67
67
66
70
69
72
77
69
70
75
69
79
--
PavYMV
76
77
74
78
76
76
67
75
72
80
78
66
70
--
26
Supplementary Table S5 - Percent identities between the deduced amino acid sequences of the CP (AV1) protein encoded by the DNA-A of the
two new begomovirus species detected in Pavonia sp. (Pavonia mosaic virus, PavMV, and Pavonia yellow mosaic virus, PavYMV) and the most
closely related viruses.
TGMV
ToSRV
ToMoLCV
ToLDV
ToYSV
SiMBoV1
SiBrV
SimMV
SiMoV
SiYNV
AbMBoV
BGMV
PavMV
PavYMV
TGMV
ToSRV
ToMoLCV
ToLDV
ToYSV
SiMBoV1
SiBrV
SimMV
SiMoV
SiYNV
AbMBoV
BGMV
PavMV
--
86
--
83
82
--
84
83
84
--
83
82
83
89
--
82
81
82
84
81
--
85
84
82
90
87
84
--
83
81
82
92
89
84
90
--
84
82
83
92
89
83
88
91
--
84
83
83
90
92
82
90
89
90
--
81
81
81
84
83
80
81
83
83
82
--
81
82
83
83
81
79
82
81
82
85
80
--
80
79
82
83
83
84
82
81
81
83
82
79
--
PavYMV
78
78
81
82
83
82
81
81
80
82
85
78
92
--
27
Supplementary Table S6 - Percent identities between the deduced amino acid sequences of the NSP (BV1) protein encoded by the DNA-B of
the two new begomovirus species detected in Pavonia sp. (Pavonia mosaic virus, PavMV, and Pavonia yellow mosaic virus, PavYMV) and the
most closely related viruses.
TGMV
TGMV
ToSRV
ToMoLCV
ToYSV
SiMBoV1
SiBrV
SimMV
AbMBoV
BGMV
PavMV
PavYMV
--
ToSRV
74
--
ToMoLCV
71
71
--
ToYSV
73
75
72
--
SiMBoV1
74
75
71
75
--
SiBrV
73
74
73
75
73
--
SimMV
AbMBoV
72
79
71
78
76
74
--
73
74
72
77
75
76
77
--
BGMV
73
73
73
76
73
74
74
73
--
PavMV
72
74
71
76
76
74
76
81
72
--
PavYMV
73
75
73
77
75
75
76
81
73
90
--
28
Supplementary Table S7 - Percent identities between the deduced amino acid sequences of the MP (BC1) protein encoded by the DNA-B of the
two new begomovirus species detected in Pavonia sp. (Pavonia mosaic virus, PavMV, and Pavonia yellow mosaic virus, PavYMV) and the most
closely related viruses.
TGMV
TGMV
ToSRV
ToMoLCV
ToYSV
SiMBoV1
SiBrV
SimMV
AbMBoV
BGMV
PavMV
PavYMV
--
ToSRV
76
--
ToMoLCV
74
73
--
ToYSV
76
76
72
--
SiMBoV1
79
78
75
76
--
SiBrV
76
74
73
74
80
--
SimMV
AbMBoV
76
83
73
77
79
76
--
78
77
75
77
81
77
78
--
BGMV
76
75
73
76
73
74
73
76
--
PavMV
79
77
75
77
81
78
77
88
76
--
PavYMV
79
77
75
77
81
79
78
89
77
94
--
29
Supplementary Figure S2 - Phylogenetic tree based on the complete DNA-B
sequences of the two new begomovirus species detected in Pavonia sp. (Pavonia
mosaic virus, PavMV, and Pavonia yellow mosaic virus, PavYMV), plus additional
sequences from New World begomoviruses. The tree was constructed by Bayesian
inference using the GTR nucleotide substitution model. Numbers at the nodes
indicate Bayesian posterior probabilities. East African cassava mosaic virus
(EACMV, an Old World begomovirus) was used as an outgroup. AbMBoV, Abutilon
mosaic Bolivia virus; BGMV, Bean golden mosaic virus; CabLCJV, Cabbage leaf
curl Jamaica virus; CoYSV, Corchorus yellow spot virus; PYMTV, Potato yellow
mosaic Trinidad virus; SiBrV, Sida Brazil virus; SiMAV, Sida mosaic Alagoas
virus; SiMBoV1, Sida mosaic Bolivia virus 1; SimMV, Sida micrantha mosaic virus;
SiYMoV, Sida yellow mottle virus; TGMV, Tomato golden mosaic virus; ToDLV,
Tomato dwarf leaf virus; ToMoLCV, Tomato mottle leaf curl virus; ToSRV, Tomato
severe rugose virus; ToYSV, Tomato yellow spot virus; ToYVSV, Tomato yellow
vein streak virus.
30
CHAPTER 2
INTRA-HOST EVOLUTION OF Tomato severe rugose virus (ToSRV)
31
Abstract
To evaluate and quantify the mutational dynamics of the bipartite begomovirus
Tomato severe rugose virus (ToSRV) in a cultivated and a non-cultivated host, plants
of tomato and Nicandra physaloides were biolistically inoculated with an infectious
clone and the leaves sampled at 30, 75 and 120 days after inoculation. Total DNA
was extracted and sequenced in the Illumina HiSeq 2000 platform. The datasets were
trimmed with the quality score limit set to 0.01, and the assembly was performed
using the infectious clone sequence as reference. We inferred high rates of nucleotide
substitution for the two DNA components in both hosts: 3.06 x 10-3 and 2.03 x 10-3
sub/site/year for the DNA-A and DNA-B, respectively, in N. physaloides, and 1.38 x
10-3 and 8.68 x 10-4 sub/site/year the for DNA-A and DNA-B, respectively, in
tomato. These values are similar to those estimated for other begomoviruses and for
viruses with single-stranded RNA genomes. Strikingly, the number of substitutions
decreased over time, with a corresponding increase in the number of identical sites,
suggesting the presence of bottlenecks during the systemic infection. In N.
physaloides, but not in tomato, stop codon suppression in the MP and NSP genes was
detected at the three time points, suggesting an adaptive strategy. Determination of
Shannon entropy indicated mutation hotspots in the N-terminal region of Rep gene,
the intergenic common region in the DNA-A and DNA-B (CR-A and CR-B,
respectively) and the long intergenic region between the MP and NSP genes in the
DNA-B (LIR-B). These results indicate that ToSRV evolves as a quasispecies, with
a high degree of genetic variability which could be partly responsible for its
prevalence in the field.
32
Introduction
The family Geminiviridae is comprised of viruses with circular, singlestranded DNA genomes encapsidated in twinned icosahedral particles. The family
includes seven genera, defined on the basis of host range (monocots or dicots), type
of insect vector (leafhoppers, treehoppers or whiteflies), genome organization
(mono- or bipartite) and phylogenetic relationships: Begomovirus, Becurtovirus,
Curtovirus, Eragrovirus, Mastrevirus, Topocuvirus and Turncurtovirus (Brown et
al., 2012; Varsani et al., 2014). The genus Begomovirus includes viruses with monoor bipartite genomes, transmitted by whiteflies (Bemisia tabaci) to dicotyledonous
plants. Begomoviruses are widespread in all tropical and subtropical regions of the
world, and cause severe diseases in a number of economically relevant crops
(Moffat, 1999; Navas-Castillo et al., 2011).
Based
on
phylogenetic
relationships
and
genome
characteristics,
begomoviruses can be divided into two groups: Old World (OW; Europe, Africa and
Asia) and New World (NW; the Americas) (Rybicki, 1994; Padidam et al., 1999b;
Paximadis et al., 1999). The majority of NW begomoviruses are bipartite, with two
genomic components named DNA-A and DNA-B. The DNA-A contains genes
involved in replication, suppression of host defenses and encapsidation of the viral
progeny. The DNA-B contains genes required for intra- and intercelular movement
in the plant and suppression of host defenses (reviewed by Rojas et al., 2005).
Populations of geminiviruses, including begomoviruses, have a high degree
of genetic variability, equivalent to that observed for viruses with single-stranded
RNA genomes (Ge et al., 2007; Duffy et al., 2008; Prasanna et al., 2010; Rocha et
al., 2013). Frequent recombination events (Padidam et al., 1999a), the occurrence of
33
pseudo-recombination between viruses with bipartite genomes (Andrade et al.,
2006), and high mutation and nucleotide substitution rates (Duffy et al., 2008; Duffy
and Holmes, 2009) are the main factors that promote the high variability observed
for begomoviruses.
Brazil is a center of genetic diversity for begomoviruses, with reports of their
detection going back to the mid-20th century (Orlando and Silberschmidt, 1946;
Costa, 1955; Flores et al., 1960). More recently, a large number of new begomovirus
species have been characterized on both cultivated and non-cultivated plants (Ribeiro
et al., 2003; Fernandes et al., 2006; Calegario et al., 2007; Ribeiro et al., 2007;
Castillo-Urquiza et al., 2008; Fernandes et al., 2008; Fernandes et al., 2009; Silva et
al., 2011; Albuquerque et al., 2012; Silva et al., 2012; Tavares et al., 2012). These
viruses, all indigenous, emerged after the introduction of the polyphagous Bemisia
tabaci Middle East-Asia Minor 1 (MEAM 1, previously know as B. tabaci biotype
B) in the mid-1990's (Ribeiro et al., 1998). The characterization of these viruses
substantiated their high degree of genetic variability (Silva et al., 2012; Lima et al.,
2013; Rocha et al., 2013). Some of these species are widely distributed throughout
the country, while others are restricted to certain regions. Tomato severe rugose virus
(ToSRV) is the predominant virus on tomato crops in southeastern and midwestern
Brazil (Fernandes et al., 2008; González-Aguilera et al., 2012).
Nicandra physaloides is a non-cultivated plant commonly found throughout
South America, including Brazil, where it is sometimes used in folk medicine (Agra
et al., 1994). Barbosa et al. (2009) found plants of N. physaloides showing symptoms
of mosaic and leaf deformation in tomato fields near the city of Sumaré in the state
of São Paulo. After cloning and sequencing, the authors identified the presence of
34
ToSRV, concluding that N. physaloides can serve as a natural reservoir for this virus
under field conditions.
The analysis of begomovirus populations found in cultivated and noncultivated plants indicates that viral populations infecting non-cultivated plants have
a higher degree of genetic variability compared to those present in cultivated plants
(Lima et al., 2013; Rocha et al., 2013). Rocha et al. (2013) found that the Brazilian
populations of begomoviruses are highly recombinant, have a rapid rate of molecular
evolution and are structured based on geographical location, which explains the
predominance of certain viral species in different regions of the country.
Until recently, all studies of genetic structure and variability of begomovirus
populations have been based on cloning and sequencing viral genomes obtained from
samples collected in the field. Although these studies have provided valuable
information, they are limited by systematic sampling, often carried out in a
fragmented way both spatially and temporally, and by the impossibility of
sequencing every genomic variant present in a given sample. In this context, the
recent advances of mass sequencing technologies (often called next generation
sequencing, or NGS) open new possibilities, allowing for example that the viral
population in a single plant be sequenced with a coverage of tens or hundreds of
thousands of times. Viral evolution could this way be followed in real time.
The wide distribution of ToSRV in the field and the high mutation rate of
begomoviruses emphasize the importance of studying the evolution of this species in
cultivated and non-cultivated hosts, to understand its evolutionary dynamics and the
emergence and establishment of new variants during a short-term viral infection.
This study aimed to track, using NGS, the evolution of a population of ToSRV in
tomato and N. physaloides.
35
Material and methods
Viral isolate
Clones corresponding to the DNA-A and DNA-B of the isolate ToSRV[BR:Pir1:05], obtained by Lima (2007), were used. Both clones correspond to 1.5
copies of the genome including two origins of replication in the same orientation.
The infectivity of the clones was confirmed by biolistic inoculation on tomato and
Nicotiana benthamiana plants.
Plant material
Tomato seeds (cv. Santa Clara) were sowed in plastic pots containing
commercial substrate (Plantmax). Seeds of Nicandra physaloides obtained from a
commercial source (AgroCosmos) were submitted to dormancy breaking by
incubating in 95% sulfuric acid for 10 minutes and rinsing with running water, and
were then planted in plastic cups containing commercial substrate (Plantmax).
Viral inoculation and sample collection
The plants were inoculated via biolistics (Aragão et al., 1996) when they
showed the first pair of true leaves. For the inoculation, 10 μg of each genomic
component were mixed with tungsten particles (M-10, Bio-Rad). Twenty plants of
each species were inoculated. Tomato plants were bombarded at 40 kgf/mmHg, and
N. physaloides plants at 50 kgf/mmHg. Two days after bombardment, the plants were
transplanted to pots containing a mixture of soil and sand in a 3:1 ratio, and
maintained in a greenhouse.
36
Infection was confirmed by extracting total DNA from systemically-infected
leaves (Doyle and Doyle, 1987), followed by rolling circle amplification (InoueNagata et al., 2004). Aliquots of the amplifications were subjected to cleavage with
the EcoRI restriction enzyme, which cleaves the ToSRV DNA-A at a single site, and
analyzed by agarose gel electrophoresis.
Sample collection started 15 days after inoculation (dai) and was performed at
15-day intervals until 120 dai. Young leaves located in the apical region of each plant
were collected and each subsequent collection was held at the subsequent node to the
previously collected branch. The process was the same for each of the two plant
species. Samples consisted of the leaves from each one of the twenty plants of each
host at each time point and were stored at -80°C until DNA extraction.
DNA extraction and sequencing
About 100 mg of leaf tissue from one of the twenty plants from each species
was used for genomic DNA extraction. The remaining 19 samples were kept for
future use, if necessary. DNA extraction was performed using the ZR Plant kit/Seed
MiniPrep DNA (Zymo Research). This kit retains DNA until 40 kpb. Next, the DNA
was purified with the DNA Clean & Concentrator-5 kit (Zymo Research), which
retains DNA until 23 kbp. After quantification, the DNA samples collected at 30, 75
and 120 dai were sent to Macrogen Inc. (Seoul, South Korea), where they were
sequenced in the Illumina HiSeq 2000 platform.
Quality control and filtering
Sequence libraries were received for bioinformatics analysis. Reads were
pair-ended with a size of 101 bases each. Quality control was initially performed
37
using the program FastQC (Andrews, 2010), to check the adapters and the quality
distribution of the bases without any prior computer processing of the libraries. Then,
the reads were trimmed using Printseq-Lite v. 0.20.4 for the first ten bases, and were
filtered for a quality score greater than 30.
Assembly and analysis of reads
The previously trimmed reads, processed to a quality score of 30, were
aligned using Geneious v. 8.0.5 assuming up to 14% mismatches. The sequence of
the ToSRV clone (2,591 nt for the DNA-A and 2,568 nt for the DNA-B) was used as
the references for alignment of the reads and for detection of nucleotide substitutions
along the viral genome.
For base polymorphism detection, an algorithm in Geneious was used to find
SNPs along the genome and in each of the coding regions in the two genomic
components. Through the output of this tool, it was possible to calculate the
frequency, to find the replacement site throughout the genome, the type of
substitution and the amino acid changed.
The total number of variations found in each analyzed region divided by the
total number of nucleotides was used to calculate the nucleotide substitution rate,
assuming that 3% of the variations were due to sequencing errors. The substitution
rate was estimated for the complete sequences of the DNA-A and DNA-B and for the
coding regions of the CP, Rep, MP and NSP genes.
The plot correlating sequence coverage with the number of mismatches was
generated in QtiPlot v. 0.9.8.10.
Shannom entropy plots were generated for each library using a script written
by Castro (2015).
38
Results
Sequence quality
Most libraries showed good sequence quality, except for the first time point
(30 dai) in tomato. We opted to filter all libraries for a score of Q30, corresponding
to an accuracy of 99.99%, and thus obtaining high homogeneity in all reads (Suppl.
Fig. S1). The FastQC analysis indicated that all data sets presented noise in the first
ten nucleotides. Thus, we chose to trim the first ten nucleotides to prevent bias in the
detection of mutations in the viral genome (Suppl. Fig. S2).
Determination of the maximum number of mismatches allowed in the assembly
Due to the bipartite nature of ToSRV, with two genomic components
containing an aprox. 200-bp common region, a strategy was necessary to avoid
incorporation of reads originating from the DNA-A in the DNA-B assembly, and
vice-versa. Assemblies were tested allowing a maximum of 4, 10, 14, 15, 20 or 25%
mismatches with no gaps, and the mapping coverage in all positions of the viral
genome was compared (Figure 1).
When up to 25% of mismatches were allowed, a considerable increase in
coverage at the common region of both genomics components was observed, without
a corresponding increase of the coverage along the remaining regions of the genome
(Figure 1). This indicated that alignment of reads from different genomic component
was taking place at the common region, with an overestimation of mutations. This
was also observed when up to 15% and 20% of mismatches were allowed (Figure 1).
On the other hand, when up to 4, 10 or 14% of mismatches were alllowed,
only small increases in coverage were observed, and the topology was the same with
no significant increase at the common region (Figure 1). For greater reliability in the
39
analysis, all sequences mapping to the common region and showing divergence from
the reference sequence were submitted to a BLASTn search to verify the genomic
component to which these reads showed higher identity.
Based on these observations, we decided to perform the genome assembly
allowing up to 14% of mismatches.
Determination of nucleotide substitutions and of the substitution rate
After mapping of all reads to the reference sequence of each genomic
component, a significantly higher coverage of the DNA-B compared to the DNA-A
was observed, together with a reduction of coverage as the viral infection progressed,
in both hosts (Table 1). This indicated that the DNA-B was present at a higher
concentration during the course of the viral infection, suggesting a higher replication
rate for the DNA-B compared to the DNA-A.
Nucleotide substitutions were quantified for the full-length DNA-A and
DNA-B as well as the CP, Rep, MP and NSP genes, allowing gaps in relation to the
reference genome (to account for insertions or deletions), up to 14% of mismatches,
and "seeds" of 18 bases. The parameters included the number of variations that
occurred in each ORF analyzed site by site, as well as the number of synonymous
and non-synonymous substitutions, the presence of stop codons, deletions and
insertions and the percentage of identical sites at each time point (30, 75 and 120 dai)
(Figure 2; Suppl. Tables S1-S4).
From the four genes analyzed, CP and Rep (both in the DNA-A) showed a
lower number of variations compared to NSP and MP (encoded by the DNA-B). All
four genes showed a consistently higher number of non-synonymous substitutions in
relation to synonymous substitutions. Samples from N. physaloides showed higher
40
number of variations when compared to tomato, for both genomic components.
Interestingly, mutations that introduced premature stop codons were observed in all
four genes, although they were more frequent in the NSP and MP genes (Figure 2;
Suppl. Tables S1-S4). The frequency ranged from 0.1% to 0.2% (mean p-value = 9.3
x 10-2) and 0.04% to 0.1% (mean p-value = 8.57 x 10-2) for the MP gene in N.
physaloides and tomato, respectively, and 0.1% to 0.3% (mean p-value = 5.52 x 10-2)
and 0.1% to 0.2% (mean p-value = 5.17 x 10-3) for the NSP gene in N. physaloides
and tomato, respectively.
In the N. physaloides samples, suppression of the regular stop codons was
observed in MP gene due to an insertion (TAA→ATA) at 30, 75 and 120 dai, and
due to a transversion (TAA→AAA) at 75 and 120 dai; in the NSP gene at 30 dai
(TAA→ATA) and 75 dai (TAA→CAA; →TTA; →TAC); and in the CP gene at 30
and 120 dai (TAA→CAA). In tomato, stop codon suppression was observed only in
the CP and MP genes, but they were deleterious over time. These mutations were not
observed in the Rep gene, in either host.
In both hosts and for all four genes, the number of variations decreased
uniformly in the genome as time passed, with a corresponding increase in the number
of identical sites (Figure 2; Suppl. Tables S1-S4).
Substitution rates were estimated for the DNA-A and DNA-B and for the
same four genes, based on the data for the 120 dai libraries (Table 2). The calculated
substitution rates were in the same order of magnitude for the DNA-A and its two
encoded genes (CP and Rep) in tomato and N. physaloides. The DNA-B and its two
encoded genes (NSP and MP) showed substitution rates one order of magnitude
higher in N. physaloides than in tomato.
41
Shannon entropy
The complexity, or diversity, of a viral population can be measured using
Shannon entropy of a sample of genomes. Nucleotide positions will have a higher
entropy if they display a high frequency of variation. Shannon entropy was
calculated for each position of the DNA-A and DNA-B at 120 dai, in the two hosts
(Figures 3 and 4). For the DNA-A, entropy values were generally low (<0.1) in
tomato. In N. physaloides, entropy values of approx. 0.4 were observed in the 5'-half
of the common region. For the DNA-B, moderate entropy values (approx. 0.3) were
observed in the short intergenic region in both tomato and N. physaloides. The high
entropy value in 3'-IR in N. physaloides is most likely due to low coverage detected
in this region.
Shannon entropy was also calculated for each host/time point to identify
mutation hotspots (Figures 5 and 6). In both hosts, the DNA-A regions encompassing
the 3'-end of the Trap gene (nt positions 1300-1500) and the middle of Rep (nt
positions 1800-2000nt) were identified as mutation hotspots (Figures 5A and 6A). In
N. physaloides, the 5'-end of the CR is a strong mutation hotspot (Figure 6A). In the
DNA-B, the two intergenic regions (nt positions 1200-1300 and 2300-2600) were
identified as hotspots for mutation (Figures 5B and 6B).
Discussion
Our experiments constitute the first attempt to calculate nucleotide
substitution rates for a geminivirus using NGS. We used a virus that has been shown
to be prevalent in tomato fields in Brazil for the last ten years (Fernandes et al., 2008;
González-Aguilera et al., 2012), and inoculated its commercial host (tomato) as well
as a host which has been shown to act as a reservoir in the field (Nicandra
42
physaloides) (Barbosa et al., 2009). Since our primary objective was to calculate
substitution rates, a deliberate choice was made to avoid any amplification of the
viral DNA (either by RCA or PCR) before sequencing. While this may have
contributed to the relatively low coverage obtained (specially for the DNA-A), it
adds confidence to the data, in terms of the observed substitutions being the result of
natural processes of mutation. A higher coverage could probably have been achieved
if an enrichment step were added to the protocol. Actually, the use of size exclusion
columns during DNA extraction was an attempt to enrich for low molecular weight
DNA (ie, viral DNA rather than plant DNA). However, it is possible that plant DNA
was fragmented and therefore was not excluded.
Geminiviruses have mutation frequencies and nucleotide substitution rates
which are similar to those observed for RNA viruses, even though they use the host's
proofreading replication machinery which should increase the fidelity of replication
(Duffy et al., 2008). Under different selection conditions, three isolates of the
mastrevirus Maize streak virus (MSV) showed a substitution rate in the order of 10-4
to 10-5 sub/site/year, suggesting that these isolates evolve in a quasispecies
organization due their high sequence heterogeneity (Isnard et al., 1998). Analyzing
the genetic variability of the monopartite begomovirus Tomato yellow leaf curl
China virus (TYLCCNV), Ge et al. (2007) reported mutation frequencies of
3.5 x 10–4 and 5.3 x 10-4 in Nicotiana benthamiana and tomato, respectively, after 60
days of infection, and also concluded that it evolves as a quasispecies. Using
Bayesian coalescence-based analysis, Duffy and Holmes (2008) found substitution
rates of 2.88 x 10-4 sub/site/year for Tomato yellow leaf curl virus (TYLCV). By
analyzing the nucleotide substitution rate of the bipartite EACMV, assuming that the
two genome segments have distinct evolutionary histories, Duffy and Holmes (2009)
43
found average rates of 1.60 x 10-3 and 1.33 x 10-4 sub/site/year for the DNA-A and
DNA-B, respectively.
Our substitution rates results are compatible with those listed above, and
consistent with the EACMV model of the two genomic components evolving at
different rates. The ToSRV DNA-A, in both tomato and N. physaloides, displayed
substitution rates similar to those reported for other members of the family
Geminiviridae. Interestingly, the substitution rate of the DNA-B and its coding
regions displayed values that were one order of magnitude higher in N. physaloides
compared to tomato. It is noteworthy that the observed values in tomato are similar
to the values found for the DNA-B of East Africa cassava mosaic virus (EACMV),
the only other bipartite begomovirus for which substitution rates were estimated
(Duffy and Holmes, 2009). It has been suggested that viruses infecting noncultivated hosts may evolve faster than those infecting cultivated hosts (Rocha et al.,
2013). It is possible that, by having a large genetic base, N. physaloides may be
capable of mounting more efficient defense responses that act specifically on the
DNA-B, placing a higher selective pressure and thus leading to the incorporation of
mutations until a variant emerges which escapes these defense responses.
In all four coding regions analyzed, nucleotide substitutions which
incorporated premature stop codons were verified. The frequency of these
substitutions was much higher in the genes encoded by the DNA-B than in those
encoded by the DNA-A. However, the frequency decreased over time. These
premature termination codons lead to the translation of truncated proteins which will,
most likely, be defective. Thus, it is possible that the DNA components harboring
these mutations are defective DNAs. By analyzing the degree of heterogeneity in
structured quasispecies populations of Hepatitis C virus (HCV) (a virus with a
44
single-stranded RNA genome), Martell et al. (1992) observed that 10% of the
sequences included premature termination codons, indicating that a significant
fraction of particles in circulation should contain defective genomes.
Interestingly, suppression of the "normal" stop codons in the MP and NSP
genes (and to a lesser extent also in the CP gene) was observed during infection in N.
physaloides. Zaccomer et al. (1995) showed that RNA viruses use suppression of
termination codons as a gene expression strategy, in addition to divided genomes,
subgenomic RNAs, frame-shifting, etc. The possible fixation of a codon for an amino
acid in place of the termination codon in the MP and NSP gene during infection in N.
physaloides may reflect the necessity of the virus to generate variability so that the
two movement proteins can interact efficiently with host factors, facilitating cell-tocell movement. Regardless, this observation suggests that these variants may present
a selective advantage. The ability to rapidly generate diversity has been proposed as
one of the reasons that make viruses capable of adapting to new ecological niches
(Roossinck, 1997).
A notable observation in our study is that the number of variations decreased
during the course of the experiment, suggesting the presence of bottlenecks during
the systemic infection. A similar observation was reported in a study where 12
experimental mutants of Cucumber mosaic virus (CMV) were inoculated in tobacco,
and a reduction of the number of mutants was observed in successive leaves as a
function of distance from the source (Li and Roossinck, 2004). However, in a study
which used NGS, an increase of Zucchini yellow mosaic virus (ZYMV) variants was
observed in successive leaves of Curcubita pepo (Dunham et al., 2014). The use of
NGS may provide greater visibility to the bottleneck phenomenon in a viral
population, as minor variants are sampled, giving better support to the analysis.
45
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49
Table 1. Total reads mapped to the Tomato severe rugose vrius (ToSRV) genome in
the tomato and Nicandra physaloides libraries at each time point (30, 75 and 120
days after inoculation, dai).
Tomato
N. physaloides
Dai
30
DNA-A
DNA-B
DNA-A
DNA-B
6.658
44.563
5.176
32.723
75
4.686
32.238
4.271
33.271
120
3.129
20.437
2.665
21.215
50
Table 2. Substitution rates in the full-length (FL) genomic components (DNA-A and DNA-B) and in the CP, Rep, NSP and MP genes of ToSRV
in (A)N. physaloides and (B) tomato.
A
Number of variations
Number of variations
(corrected)
Nucleotides sequenced
Substitution rate
(sub/site/year)
Substitution rate
(estimated) (sub/site/year)
FL DNA-A
254
246.38
CP
33
32.01
Rep
57
55.29
FL DNA-B
1,333
1293.01
NSP
254
246.38
MP
267
258.99
245,106
1.01 x 10-3
64,546
5.0 x 10-4
91,860
6.02 x 10-4
1,933,133
6.69 x 10-4
531,472
4.64 x 10-4
700,699
3.7 x 10-4
3.06 x 10-3
1.51 x 10-3
1.83 x 10-3
2.03 x 10-3
1.41 x 10-3
1.12 x 10-3
FL DNA-A
CP
Rep
FL DNA-B
NSP
MP
134
129.98
26
25.22
43
41.71
548
531.56
144
139.68
157
152.29
287,330
4.52 x 10-4
74,776
3.37 x 10-4
108,764
3.84 x 10-4
1,862,335
2.85 x 10-4
527,879
2.65 x 10-4
699,655
2.17 x 10-4
1.38 x 10-3
1.03 x 10-3
1.17 x 10-3
8.68 x 10-4
8.05 x 10-4
6.62 x 10-4
B
Number of variations
Number of variations
(corrected)
Nucleotides sequenced
Substitution rate
(sub/site/year)
Substitution rate
(estimated) (sub/site/year)
51
Figure legends
Figure 1. Coverage of the viral genome assuming different values of mismatches.
Figure 2. Variability observed in the ToSRV genes (CP, Rep, MP, NSP) after
biolistic inoculation of tomato and Nicandra physaloides. (A) Total number of
variations, (B) Percentage of identical sites, (C) Total number of synonymous
substitutions, (D) Total number of non-synonymous substitutions. dai, days after
inoculation.
Figure 3. Shannon entropy for the ToSRV DNA-A (A) and DNA-B (B) in tomato at
120 days after inoculation.
Figure 4. Shannon entropy for the ToSRV DNA-A (A) and DNA-B (B) in Nicandra
physaloides at 120 days after inoculation.
Figure 5. Shannon entropy for the ToSRV DNA-A (A) and DNA-B (B) at each time
point (30, 75 and 120 days after inoculation) in tomato.
Figure 6. Shannon entropy for the ToSRV DNA-A (A) and DNA-B (B) at each time
point (30, 75 and 120 days after inoculation) in Nicandra physaloides.
Supplementary Figure S1. Box-plots of the quality of bases and their positions in
the reads for the sample of N. physaloides collected 30 days after inoculation. On the
52
left, reads R1 (+) and on the right, reads R2 (-). (A) Unprocessed for filtering quality,
(B) processed for filtering quality for score Q30.
Supplementary Figure S2. Sequence content among reads for the sample of N.
physaloides with 30 dai. Above, reads R1 (+) without trimming of the first ten
nucleotides and below, reads R1 after trimming of the first ten nucleotides.
53
Figure 1
54
Figure 2
A
C
B
D
55
Figure 3
A
B
56
Figure 4
A
B
57
Figure 5
A
B
58
Figure 6
A
B
59
Supplementary Table S1. Variability observed in the ToSRV CP gene after
biolistic inoculation of tomato and Nicandra physaloides, at 30, 75 and 120 days
after inoculation (dai).
N. physaloides
Total number of
variations
Synonymous
substitutions
Non-synonymous
substitutions
Transitions
Transversions
Deletions
Insertions
Stop codons
% identical sites
Tomato
30 dai
75 dai
120 dai
30 dai
75 dai
120 dai
69
126
33
57
42
26
12
20
8
19
10
5
57
19
20
1
2
2
87.6
106
31
34
5
2
6
76.8
25
12
13
3
0
2
94.8
38
22
20
2
1
1
91
32
16
17
3
0
0
93.4
21
8
13
2
0
2
96.2
60
Supplementary Table S2. Variability observed in the ToSRV Rep gene after
biolistic inoculation of tomato and Nicandra physaloides, at 30, 75 and 120 days
after inoculation (dai).
N. physaloides
Total number of
variations
Synonymous
substitutions
Non-synonymous
substitutions
Transitions
Transversions
Deletions
Insertions
Stop codons
% identical sites
Tomato
30 dai
75 dai
120 dai
30 dai
75 dai
120 dai
144
139
57
68
93
43
20
17
16
11
15
10
124
30
54
6
4
4
82.8
122
28
46
2
0
6
82
41
21
15
2
2
1
92.7
57
18
15
1
0
4
89.6
78
24
27
7
0
2
87.7
33
15
13
2
0
1
94.7
61
Supplementary Table S3. Variability observed in the ToSRV NSP gene after
biolistic inoculation of tomato and Nicandra physaloides, at 30, 75 and 120 days
after inoculation (dai).
N. physaloides
Total number of
variations
Synonymous
substitutions
Non-synonymous
substitutions
Transitions
Transversions
Deletions
Insertions
Stop codons
% identical sites
Tomato
30 dai
75 dai
120 dai
30 dai
75 dai
120 dai
451
424
254
271
237
144
69
75
42
48
45
27
382
125
151
14
7
23
49.9
349
132
147
18
5
15
52.1
212
84
96
10
3
16
67.2
223
101
103
3
3
12
68.6
192
91
87
14
5
12
75.2
117
58
46
7
0
4
86.3
62
Supplementary Table S4. Variability observed in the ToSRV MP gene after
biolistic inoculation of tomato and Nicandra physaloides, at 30, 75 and 120 days
after inoculation (dai).
N. physaloides
Total number of
variations
Synonymous
substitutions
Non-synonymous
substitutions
Transitions
Transversions
Deletions
Insertions
Stop codons
% identical sites
Tomato
30 dai
75 dai
120 dai
30 dai
75 dai
120 dai
480
466
267
408
326
157
77
79
54
69
61
40
403
149
151
23
11
387
131
208
14
9
213
82
123
8
2
339
143
158
13
5
265
121
144
15
2
117
63
72
8
0
29
47.6
20
49.8
15
68.7
19
55.7
16
63.3
5
81.4
63
Supplementary Figure S1
A
B
64
Supplementary Figure S2
65